Separation of fibrinogen from plasma proteases

ABSTRACT

The present invention relates to methods for purifying fibrinogen. In one aspect, the present invention relates to a method of separating fibrinogen from plasma fraction I precipitate. In another aspect, the invention relates to the purification of fibrinogen using ion exchange chromatography.

This application is a 371 of PCT/AU00/01585 and claims foreign priorityto Australian application PQ 4842, filed 23 Dec. 1999, and Australianapplication PQ 4841, filed filed 23 Dec. 1999.

FIELD OF THE INVENTION

The present invention relates to methods for purifying fibrinogen. Inone aspect, the present invention relates to a method of separatingfibrinogen from plasma fraction I precipitate. In another aspect, theinvention relates to the purification of fibrinogen using ion exchangechromatography.

BACKGROUND OF THE INVENTION

The isolation of human fibrinogen has traditionally been carried out byclassical plasma fractionation methods. Fibrinogen is precipitated fromplasma either with ethanol (Blomback and Blomback, 1956), ammoniumsulphate (Takeda, 1996), β alanine/glycine (Jakobsen and Kieruif, 1976),polymers (polyethelene glycol) and low ionic strength solutions (Holm,1985) with relative high yield and homogeneity.

Further purification of fibrinogen precipitates can be achieved byion-exchange chromatography conditions (Stathakis et al, 1978) andaffinity chromatography (Kuyas et al, 1990). Specific contaminants canbe absorbed out for example fibronectin on immobilised gelatine andplasminogen an immobilised lysine (Vuento et al, 1979).

Precipitation methods are widely used for the manufacture of commercialfibrinogen. Chromatographic methods are now being explored as analternative or to improve the purity of fibrinogen concentrates.

WO 99/37680 describes a method for the large scale separation offibrinogen from other blood proteins in human blood plasma. The processinvolves the use of a heparin precipitated paste as a starting materialfor the purification of fibrinogen. The heparin precipitated paste is aby-product from the manufacturing process of Factor VIII(Antihaemophilic Factor, AHF).

Attempts to produce fibrinogen free of plasminogen or to purifyplasminogen itself have been widely published in the literature. Themost common method is to utilise the ability of lysine to bind to one ofthe two “kringles” in the plasminogen molecule. The use of affinitychromatography step was first disclosed in a paper published by Deutschand Mertz in 1970. Baxter International Inc. utilised this technology,which incorporated the use of lysine-sepharose material in a dedicatedstep to remove plasminogen from their fibrinogen as disclosed in thepatent WO 95/25748 for the large scale manufacture of a fibrinogenconcentrate free of destabilising levels of plasminogen product. Othertechniques published in the scientific literature again utilise thebinding of either lysine or ε-amino caproic acid. However, they areemployed to alter the solubility of the plasminogen molecule. Followingthe addition of lysine to a dilute fibrinogen solution, the subsequentsolution is then precipitated in the presence of 7% ethanol. Removal ofplasminogen is stated at greater than 90% with a repeat of the stepleading to total removal of the contaminant (Mosesson, 1962).Precipitation methods are widely used for the manufacture of commercialfibrinogen, however, the work published by Mosesson (1962) relys on adilute solution of fibrinogen which is not a practical process forimplementation at a production scale.

The use of ion-exchange chromatography and ε-amino caproic acid to bindand elute plasminogen independent of pH or ionic strength was disclosedin a patent (WO 94/00483) lodged in 1994 by Novo Nordisk A/S describingthe purification of kringle containing proteins. This method choosesS-sepharose as the resin of choice. Also, a combination of gelfiltration and ion-exchange chromatography has been utilised to purifyplasminogen. (Robbins et al, 1965).

SUMMARY OF THE INVENTION

The present inventors have now found that fibrinogen may be recovered ina purified form from a starting material consisting of Fraction I paste.The fibrinogen recovered in this process is free of destabilising levelsof plasminogen and other proteases. Fibrinogen recovered in this manneralso contains factor XIII, which is required to enhance thecross-linking of fibrin polymers in the production of fibrin glue.Furthermore, the yields of fibrinogen obtained by this process areunexpectedly higher than those obtained in methods which use alternativestarting materials, such as heparin precipitated paste.

The present inventors have also developed an improved method forrecovering fibrinogen from an ion-exchange column which involves theaddition of at least one ω-amino acid to the fibrinogen-containingmaterial applied to the column or to the solution used to wash thecolumn prior to elution of the fibrinogen.

When used herein, the phrase “Fraction I precipitate” refers to frozenplasma which has been thawed and the cryoprecipitate removed bycentrifugation. The resultant cryosupernatant is then mixed with ethanolto precipitate Fraction I.

Accordingly, in a first aspect the present invention provides a methodof purifying fibrinogen, which method comprises extracting fibrinogenfrom a Fraction I precipitate by admixing the Fraction I precipitatewith an extraction buffer such that fibrinogen is solubilised in theextraction buffer, wherein the extraction buffer comprises salt at aconcentration of at least 0.1M and heparin at a concentration of atleast 10 IU/ml.

In a preferred embodiment of the first aspect, the concentration of saltis at least 0.2M, more preferably at least 0.4M, more preferably about0.8M.

In a further preferred embodiment of the first aspect, the extractionbuffer comprises at least one salt selected from the group consisting ofchloride, phosphate and acetate salts, and more preferably comprisesNaCl.

In a further preferred embodiment of the first aspect, the extractionbuffer also comprises Tri-sodium citrate at a concentration of about 20mM.

In a further preferred embodiment of the first aspect, the extractionbuffer further comprises at least one ω-amino acid. Preferably, the atleast one ω-amino acid is present in the extraction buffer at aconcentration of at least 5 mM.

In a further preferred embodiment of the first aspect, the extractionbuffer comprises antithrombin III (ATIII) at a concentration of at leastabout 1 IU/ml.

In a further preferred embodiment of the first aspect, the extractionbuffer comprises Tri-sodium citrate at a concentration of about 20 mM,NaCl at a concentration of about 0.8M, heparin at a concentration ofabout 60 IU/ml and at least one ω-amino acid at a concentration of about5 mM. Preferably the extraction buffer has a pH of about 7.3.

In a further preferred embodiment of the first aspect, the extraction offibrinogen is performed at about 37° C. Preferably, the extraction isperformed for at least 60, more preferably at least 90 minutes.

In a further preferred embodiment of the first aspect, the methodfurther comprises the step of incubating the extracted fibrinogensolution with aluminium hydroxide followed by centrifugation and removalof the precipitate.

In a further preferred embodiment of the first aspect, the methodfurther comprises the step of precipitating the fibrinogen from theextracted fibrinogen solution by the addition of a glycine saline.(Gly/NaCl) buffer. Preferably, the Gly/NaCl buffer comprises glycine ata concentration of around 2.1M, Na-citrate at a concentration of around20 mM, sodium chloride at a concentration of around 3.6M and CaCl₂ at aconcentration of around 2.4 mM.

In a further preferred embodiment of the first aspect, the methodfurther comprises the step of resolubilising the fibrinogen precipitatein a buffer comprising NaCl at a concentration of around 100 mM, CaCl₂at a concentration of around 1.1M, Na-citrate at a concentration ofaround 10 mM, tris at a concentration of around 10 mM and sucrose at aconcentration of around 45 mM, preferably with a pH of about 6.9.

In a further preferred embodiment of the first aspect, the methodfurther comprises the steps of:

applying the extracted fibrinogen solution to an ion exchange matrixunder conditions such that fibrinogen binds to the matrix;

eluting the fibrinogen from the matrix; and

optionally recovering the fibrinogen from the eluate.

In a further preferred embodiment of the first aspect, the methodfurther comprises washing the ion exchange matrix with a buffercomprising at least one ω-amino acid prior to eluting the fibrinogenfrom the matrix. Preferably the wash buffer comprises the at least oneω-amino acid at a concentration of at least 5 mM.

In a further preferred embodiment, the wash buffer comprises (i) tris ata concentration of about 50 mM, (ii) at least one ω-amino acid at aconcentration of about 20 mM, and NaCl at a concentration of about 90mM. Preferably, the buffer has a pH of about 8.0. Preferably, the bufferhas a conductivity of about 11.1 mS/cm.

In a second aspect, the present invention provides a method of purifyingfibrinogen, which method comprises:

-   -   (a) extracting fibrinogen from a Fraction I precipitate by        admixing the Fraction I precipitate with an extraction buffer        such that fibrinogen is solubilised in the extraction buffer,        wherein the extraction buffer comprises salt at a concentration        of at least 0.1M;    -   (b) precipitating the fibrinogen; and    -   (c) solubilising the fibrinogen in a solution comprising at        least one ω-amino acid at a concentration of at least 100 mM.

In a preferred embodiment of the second aspect, the concentration ofsalt in the extraction buffer is at least 0.2M, more preferably at least0.4M, more preferably about 0.8M.

In a further preferred embodiment of the second aspect, the extractionbuffer comprises at least one salt selected from the group consisting ofchloride, phosphate and acetate salts, and more preferably comprisesNaCl.

Preferably, the extraction buffer also comprises Tri-sodium citrate at aconcentration of about 20 mM.

In a preferred embodiment of the second aspect, the extraction bufferfurther comprises heparin at a concentration of at least 10 IU/ml, morepreferably about 60 IU/ml.

In a further preferred embodiment of the second aspect, the extractionbuffer further comprises at least one ω-amino acid. Preferably, the atleast one ω-amino acid is present in the extraction buffer at aconcentration of at least 5 mM.

In a further preferred embodiment of the second aspect, the extractionbuffer comprises Na-citrate at a concentration of about 20 mM, NaCl at aconcentration of about 0.8M and heparin at a concentration of about 60IU/ml. Preferably the extraction buffer has a pH of about 7.3.

In a further preferred embodiment of the second aspect, the fibrinogenis precipitated in step (b) by the addition of a glycine saline(Gly/NaCl) buffer. Preferably, the Gly/NaCl buffer comprises glycine ata concentration of around 2.1M, Na-citrate at a concentration of around20 mM, sodium chloride at a concentration of around 3.6M and CaCl₂ at aconcentration of around 2.4 mM.

In a further preferred embodiment of the second aspect, the fibrinogenprecipitate is solubilised in step (c) using a buffer comprising NaCl ata concentration of around 100 mM, CaCl₂ at a concentration of around1.1M, Na-citrate at a concentration of around 10 mM, tris at aconcentration of around 10 mM and sucrose at a concentration of around45 mM. Preferably, the buffer has a pH of about 6.9.

In a further preferred embodiment of the second aspect, the methodfurther comprises:

(d) applying the fibrinogen solution from step (c) to an ion exchangematrix under conditions such that fibrinogen binds to the matrix;

(e) eluting the fibrinogen from the matrix; and

(f) optionally recovering the fibrinogen from the eluate.

In a preferred embodiment of the second aspect, the method furthercomprises washing the ion exchange matrix with a buffer comprising atleast one ω-amino acid prior to eluting the fibrinogen from the matrix.Preferably the wash buffer comprises the at least one ω-amino acid at aconcentration of at least 5 mM.

In a further preferred embodiment of the second aspect, the wash buffercomprises (i) tris at a concentration of about 50 mM, (ii) at least oneω-amino acid at a concentration of about 20 mM, and NaCl at aconcentration of about 90 mM. Preferably, the buffer has a pH of about8.0. Preferably, the buffer has a conductivity of about 11.1 mS/cm.

In a further preferred embodiment of the second aspect, the fibrinogencontaining solution (preferably comprising the ω-amino acid) is dilutedsuch that the conductivity is below 10.5 mS/cm before it is applied tothe ion exchange matrix.

In a further preferred embodiment of the second aspect, the fibrinogenis eluted from the matrix in a buffer comprising about 10 mM Tris, 10 mMcitrate, 45 mM sucrose; and NaCl at a concentration of between 200 mM to1.0M, more preferably about 400 mM. Preferably, the buffer has a pH ofabout 7.0.

In a third aspect the present invention provides a method of purifyingfibrinogen, which method comprises:

(a) extracting fibrinogen from a fibrinogen containing material byadmixing the material with an extraction buffer such that fibrinogen issolubilised in the extraction buffer, wherein the extraction buffercomprises at least one ω-amino acid at a concentration of at least 5 mM;

(b) applying the extraction buffer from step (a) to an ion exchangematrix under conditions such that fibrinogen binds to the matrix;

(c) eluting the fibrinogen from the matrix; and

(d) optionally recovering the fibrinogen from the eluate.

In a preferred embodiment of the third aspect, the method furthercomprises washing the ion exchange matrix after step (b) with a solutioncomprising at least one ω-amino acid.

In a fourth aspect the present invention provides a method of purifyingfibrinogen from a fibrinogen containing solution which method comprises:

(a) applying the solution to an ion exchange matrix, under conditionssuch that fibrinogen binds to the matrix;

(b) washing the ion exchange matrix with a solution comprising at leastone ω-amino acid;

(c) eluting the fibrinogen from the matrix; and

(d) optionally recovering the fibrinogen from the eluate.

In a preferred embodiment of the fourth aspect, the method furthercomprises adding at least one ω-amino acid to the solution beforeapplying to the ion exchange matrix.

In the context of the third and fourth aspects of the present invention,the fibrinogen containing material may be any material derived fromplasma which includes fibrinogen. Examples of such solutions include,but are not limited to, plasma (including anti-coagulated plasma), orplasma fractions. In preferred embodiment, the material is a heparinprecipitated paste, which is a by-product in the manufacturing processof Factor VIII. The heparin precipitated paste may be solubilised with asalt solution to provide a fibrinogen preparation of high specificactivity. A process for precipitating fibrinogen from a cryoprecipitateextract using heparin as described in Winkelman et al. 1989, the entirecontents of which are incorporated herein by reference. Alternatively,the fibrinogen containing material is extracted from Fraction 1precipitate, preferably in accordance with a method of the first orsecond aspects of the present invention.

In a further preferred embodiment of the third or fourth aspects, theω-amino acid is present in the extraction buffer at a concentration ofbetween 5-500 mM, more preferably between 50-500 mM, and more preferablyaround 100 mM.

In a further preferred embodiment of the third or fourth aspects, thefibrinogen containing solution (preferably comprising the ω-amino acid)is diluted such that the conductivity is below 10.5 mS/cm before it isapplied to the ion exchange matrix.

In a further preferred embodiment of the third or fourth aspects, thebuffer used to wash the ion exchange matrix comprises (i) tris at aconcentration of about 50 mM, (ii) a ω-amino acid at a concentration ofabout 20 mM, and NaCl at a concentration of about 90 mM. Preferably, thebuffer has a pH of about 8.0. Preferably, the buffer has a conductivityof about 11.1 mS/cm.

In a further preferred embodiment of the third or fourth aspects, thefibrinogen is eluted from the matrix in a buffer comprising about 10 mMTris, 10 mM citrate, 45 mM sucrose; and NaCl at a concentration ofbetween 200 mM to 1.0M, more preferably about 400-500 mM. Preferably,the buffer has a pH of about 7.0.

In a preferred embodiment of the first, second, third or fourth aspectsof the present invention, the ω-amino acid contains at least 4 carbonatoms in the carbon chain between the carboxylic acid and the ω-aminogroup. The carbon chain may be linear or cyclic. Examples of suitablelinear ω-amino acids are 4-aminobutyric acid, 5-aminopentoic acid,6-aminohexanoic acid (ε-amino caproic acid (EACA)), 7-aminoheptanoicacid, 8-aminooctanoic acid, and arginine. Examples of cyclic ω-aminoacids are trans-4-aminomethyl cyclohexane carboxylic acid (tranexamicacid) and para-aminomethyl benzoic acids. In a particularly preferredembodiment, the ω-amino acid is EACA.

Ion exchange matrices are known in the art and any suitable matrix maybe used in the present invention. A preferred matrix is the MacroPrep HQResin (BioRad, catalaogue no. 156-0041). In a further preferredembodiment, the ion exchange matrix is loaded into a column.

It will be appreciated by those skilled in the art that the methods ofthe third and fourth aspects have the potential to provide analternative to affinity chromatography for the large scale production offibrinogen free of destabilising levels of plasminogen and otherproteases. The methods in these aspects require only a single processingstep using ion exchange chromatography for the isolation of fibrinogenfree of destabilising levels of plasminogen and other proteases frombiological fluids with a high recovery rate (approximately 75%). The useof this novel method for the purification of fibrinogen from bloodproteins has the potential to enable a simpler method of manufactureleading to a product which is superior in both purity and stability.

The technology of the current invention offers many advantages withregards to both the manufacture of fibrinogen and the use of fibrinogenin a fibrin sealant product. The removal of plasminogen from thefibrinogen component allows the manufacturer the liberty of not havingto add inhibitory agents, either human, animal or synthetically derived,in order to obtain the desired stability of the fibrinogen component andfibrin glue. Addition of inhibitory agents can lend itself to otherproblems which are avoided by the removing plasminogen from the finalproduct.

Finally, the production costs of an ion-exchange resin is far moreeconomical than the cost of lysine-sepharose or immobilised lysine resinwhich are used in affinity chromatography procedures.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Abbreviations used herein: TP Total Protein CP Clottable protein FXIIIFactor XIII FII Factor II Plasm. Plasminogen FN Fibronectin ATIIIAntithrombin III F1P Fraction 1 paste SFP Solubilised fraction 1 pasteASFP Alhydrogel absorbed solubilised fraction 1 paste GASFPResolubilised Gly/NaCl precipitated solubilised fraction 1 pasteSDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresisGly/NaCl Glycine/Saline SD Solvent/detergent εACA epsilon aminocaproicacid TNBP Tri-N-butyl phosphate Al(OH)₃ Aluminium hydroxide RT Roomtemperature PET Plasma Engineering Technology IEX Ion exchange SHPSolubilised heparin paste supernatant

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Yield of clottable fibrinogen obtained in paste to buffer ratiostudy (I)

FIG. 2: Yield of total fibrinogen obtained in paste to buffer ratiostudy (1)

FIG. 3: Yield of total fibrinogen obtained in paste to buffer ratiostudy (2)

FIG. 4: Yield of clottable fibrinogen obtained over time duringextraction of Fraction I paste.

FIG. 5: Effect of temperature on extraction of fibrinogen from FractionI paste.

FIG. 6: Flow chart depicting a preferred fibrinogen purification processincorporating the ion-exchange chromatography method of the presentinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Extractionof Fibrinogen from Fraction 1 Precipitate

1.1 Materials and Methods

1.1.1 Heparin Paste Extraction Procedure

Fraction I paste is extracted at a ratio of 1 g:8.33 g heparin pasteextraction buffer unless stated otherwise. The extraction is performedat room temperature for 2 hours.

1.1.2 Heparin Paste Extraction Buffer

-   0.4 M NaCl,-   5 mM εACA,-   20 mM Na-citrate,-   pH 7.3.    1.1.3 Alhydrogel Absorption

A solution of 2% aluminium hydroxide Al(OH)₃, also known as alhydrogel,is added to solubilised heparin paste superntant (SHP) at aconcentration of 10.8%. The mixture is incubated with stirring for 15minutes at room temperature, centrifuged for 10 minutes, and the pelletdiscarded.

1.1.4 Gly/NaCl Precipitation

The alhydrogel supernatant (ASFP) and Gly/NaCl buffer are heated to 30°C.±3° C. The supernatant is then added to the Gly/NaCl buffer, over 4.5minutes, at a ratio of 1:2.05. The supernatant is then incubated at 30°C. with stirring for 20 mins, before centrifuging for 10 mins at 5010 g.The supernatant is discarded and the precipitate resolubilised using avolume of Buffer D equal to one third of the mass of the supernatantobtained after extraction of fibrinogen from Fraction I paste. Theprecipitate may be stirred at room temperature during resolubilisation.

1.1.5 Gly/NaCl Buffer

-   2.1M glycine-   20 mM Na-citrate-   3.6M NaCl-   2.4 mM CaCl₂    1.1.6 Buffer D-   100 mM NaCl-   1.1 mM CaCl₂-   10 mM Na-citrate-   10 mM tris-   45 mM sucrose-   pH 6.9    1.1.7 Solvent Detergent Treatment

Solvent detergent treatment is performed by adding 1% polysorbate 80 and0.3% TNBP to the resolubilised Gly/NaCl precipitate (GASFP).

1.1.8 Wet Heat Treatment

Solvent detergent treated fibrinogen is diluted 1/15 with concentratedsucrose/glycine buffer to a final concentration of approximately 1 mg/mLprotein, 60% sucrose and 1 M glycine. The formulated product is heatedto 60° C. and incubated for 10 hours.

1.1.9 Ion Exchange Chromatography

Wet heat treated fibrinogen is applied to an equilibrated anion exchangeresin. After washing of the resin, the product is eluted using asalt-containing buffer.

1.1.10 Stability at 37° C.

In process samples were incubated at 37° C. in a water bath and samplestaken and frozen at regular time intervals. The stability samples wereanalysed by SDS-PAGE under reducing conditions. Stability was assessedqualitatively as the last time point where no degradation of the αsubunit of fibrinogen was observable by eye on the gel.

1.1.11 Extraction 1 Procedure

Fraction 1 paste was obtained fresh from production. 6 g was extractedimmediately using heparin paste extraction buffer. The solubilisedfraction 1 paste was then aliquoted and stored frozen at −80° C. untilassayed.

1.1.12 Extraction 2 Procedure

Fraction 1 paste was obtained fresh from production. 12 g was extractedimmediately using heparin paste extraction buffer. The paste was treatedwith Al(OH)₃ and then precipitated using Gly/NaCl buffer. Theprecipitate was resolubilised in Buffer D. Samples were taken at eachstage and frozen at −80° C. until assayed. Remaining fraction 1 pastewas stored at −80° C.

1.1.13 Extraction 3 (Frozen Paste) Procedure

Fraction 1 paste (30 g) was thawed at 37° C. and extracted using heparinpaste extraction buffer. The solubilised fraction 1 paste was treatedwith Al(OH)₃ and precipitated using Gly/NaCl buffer. The Gly/NaClprecipitate was then resolubilised using Buffer D. εACA was spiked intosamples of the resolubilised Gly/NaCl precipitate at concentrations of 0mM, 20 mM, 100 mM, 200 mM and 500 mM. The samples were then assessed forstability at 37° C.

Resolubilised Gly/NaCl precipitate was treated with SD and applied tothe MacroPrep HQ ion exchange column. Fractions were collected and alsoassessed for stability at 37° C.

1.1.14 Extraction 4 Procedure

Fraction 1 paste was obtained fresh from production. 40 g was extractedimmediately using heparin paste extraction buffer. After 2 hours ofextraction, the fraction 1 paste was not completely solubilised. Thematerial was centrifuged* and the supernatant (solubilised fraction 1paste #1) treated with Al(OH)₃ and Gly/NaCl precipitated. The Gly/NaClprecipitate was resolubilised using Buffer D. εACA was spiked into 2sets of samples of the resolubilised Gly/NaCl precipitate atconcentrations of 0 mM, 20 mM, 125 mM, 250 mM and 500 mM. One group ofsamples was incubated at 37° C. immediately and assessed for stabilityover time. The other group of samples was stored frozen at −80° C. for60 hours, thawed and then incubated at 37° C. for stability. Theremaining resolubilised Gly/NaCl precipitate was SD treated and appliedto the ion exchange column.

*The non-solubilised fraction 1 material (14.13 g) was then re-extractedin heparin paste extraction buffer containing 0.8 M NaCl. Thesolubilised fraction 1 material (#2) was aliquoted and stored frozen at−80° C.

1.1.15 Addition of ATIII to Extraction Buffer

Fraction 1 paste was obtained from production and half was stored for4.5 days at 4° C. and the other half at −80° C. In this experiment,extractions were performed using the 4° C. (fresh paste) and the −80° C.(frozen paste) extracted in buffer with and without 1 IU/mL ATIII. Anadditional change to the standard extraction buffer was the increase insalt concentration to 0.8 M.

Fraction 1 paste (6 g) was extracted under each of the followingconditions:

-   -   (1) Fresh paste extracted in 20 mM NaCitrate, 5 mM εACA, 0.8M        NaCl, pH 7.3.    -   (2) Fresh paste extracted in 20 mM NaCitrate, 5 mM εACA, 0.8M        NaCl, 1 IU/mL ATIII, pH 7.3.    -   (3) Frozen paste thawed at 37° C. and then extracted in 20 mM        NaCitrate, 5 mM εACA, 0.8M NaCl, pH 7.3.    -   (4) Frozen paste thawed at 37° C. and then extracted in 20 mM        NaCitrate, 5 mM εACA, 0.8M NaCl, 1 IU/mL ATIII, pH 7.3.

The solubilised fraction 1 paste was subjected to alhydrogel absorptionand precipitated using Gly/NaCl buffer. The precipitates were then splitin half. Half the precipitate was stored at −80° C. and the other halfwas resolubilised using Buffer D. The resolubilised precipitate was thensplit in half again and 0 εACA or 250 mM εACA was added. The sampleswere then assessed for stability at 37° C.

The frozen Gly/NaCl ppt was thawed at 37° C. and resolubilised usingBuffer D+100 mM εACA (warmed to 30° C.). Resolubilisation was performedat 30° C. The samples were then assessed for stability at 37° C.

1.1.16 Addition of Heparin to Extraction Buffer

Fraction 1 paste was obtained from production after storage for 3 daysat 4° C. In this experiment, 4 extractions were performed using bufferwith and without 1 IU/mL ATIII in the presence of 20 IU/mL or 60 IU/mLheparin.

Fraction 1 paste (6 g) was extracted under each of the followingconditions:

-   -   (1) Fresh paste extracted in 20 mM NaCitrate, 5 mM εACA, 0.4 M        NaCl, 20 IU/mL heparin pH 7.3.    -   (2) Fresh paste extracted in 20 mM NaCitrate, 5 mM εACA, 0.4 M        NaCl, 60 IU/mL heparin, pH 7.3.    -   (3) Fresh paste extracted in 20 mM NaCitrate, 5 mM εACA, 0.4 M        NaCl, 20 IU/mL heparin, 1 IU/mL ATM, pH 7.3.    -   (4) Fresh paste extracted in 20 mM NaCitrate, 5 mM εACA, 0.8 M        NaCl, 60 IU/mL heparin, 1 IU/mL ATIII, pH 7.3.

The solubilised fraction 1 paste was treated with Al(OH)₃ andprecipitated using Gly/NaCl buffer. The precipitates were then split inhalf. Half the precipitate was stored at −80° C. and the other half wasresolubilised using Buffer D. The resolubilised precipitate was thensplit in half again and 0 εACA or 250 mM εACA was added. The sampleswere then assessed for stability at 37° C.

Resolubilisation of the frozen pellet was performed by the addition ofBuffer D+100 mM εACA (warmed to 30° C.) into the frozen Gly/NaClprecipitates. Resolubilisation was performed at 30° C. The samples werethen assessed for stability at 37° C.

1.1.17 Paste: Buffer Ratio Study

Study I:

Fraction 1 paste was obtained fresh from production. 1.5 g, 3 g, 4.5 g,6 g, 7.5 g & 9 g were resolubilised in 50 g of extraction buffercontaining 0.8 M NaCl. Samples were taken for total and clottableprotein. Remaining material was discarded.

Study II:

Fraction 1 paste (Batch # 3715001253) was obtained fresh fromproduction. 4.5 g, 9 g, 13.5 g, 18 g, 22.5 g & 27 g were resolubilisedin 150 mL of extraction buffer containing 0.8 M NaCl, 60 IU/mL heparinat 37° C. for 90 minutes. Samples were taken for total and clottableprotein.

1.1.18 Extraction Temperature Study

Fraction 1 paste was obtained fresh from production. 18 g was extractedin 150 mL of 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, pH 7.3 for 2 hoursat room temperature. Another 18 g was extracted in 150 mL of 20 mMNaCitrate, 5 mM εACA, 0.8 M NaCl, pH 7.3 for 2 hours at 37° C. Sampleswere taken at 30 min., 60 min., 90 min. and 120 min. throughout theextraction for total and clottable protein. The solubilised paste wasthen centrifuged and another sample taken.

1.1.19 Production Scale Extraction Study

Production Scale Extraction I:

Fraction 1 paste was obtained fresh from production. 20.0 kg wasextracted by PET group in 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, 60IU/mL heparin, pH 7.3 at a ratio of 1 g paste:8.33 g buffer. Extractionwas performed at 37° C. for 90 min.

Solubilised fraction 1 paste was then subjected to alhydrogel absorptionand Gly/NaCl precipitation were performed. The precipitate was split intwo and half the precipitate resolubilised in Buffer D containing 100 mMεACA and the other half frozen at −80° C. The resolubilised Gly/NaClprecipitate was then treated with SD, wet heat treated and applied tothe ion exchange column. The eluate was collected, sampled and frozen at−80° C.

Resolubilisation of the frozen pellet was performed by the addition ofBuffer D+100 mM εACA (warmed to 30° C.) into the frozen Gly/NaClprecipitates. Resolubilisation was performed at 30° C. The samples werethen assessed for stability at 37° C.

Production Scale Extraction II:

Fraction 1 paste was obtained fresh from production. 30.0 kg wasextracted by PET group in 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, 60IU/mL heparin, pH 7.3 at a ratio of 1 g paste 8.33 g buffer. Extractionwas performed at 37° C. for 90 min. Samples were taken for total andclottable protein.

1.2 Results

1.2.1 Extraction 1 Procedure

Fraction 1 paste (6 g) was solubilised in 50 mL extraction buffer.Following centrifugation, 52.47 g supernatant was collected and thepellet discarded.

Protein Characterisation

The solubilised fraction 1 paste was assayed for total protein,clottable protein, factor XIII, plasminogen and fibronectin as detailedin Table 1. The yield of fibrinogen per kilogram of plasma is calculatedin Table 2. Size exclusion analysis was performed using Superose 6, theresults of which are detailed in Table 3.

TABLE 1 Protein characterisation of solubilised fraction 1 paste TotalSFP Protein Fibrinogen fibrinogen % CP FXIII Plasminogen FN Sample (g)(mg/mL) (mg/mL) (mg) (%) (IU/mL) (μg/mL) (mg/mL) SFP 52.7 26.56 17.20902.5 65 13.57 125-129 0.44

The characterisation of the solubilised fraction 1 paste shows that highlevels of protein are extracted of which approximately 65% is clottableprotein or fibrinogen. The solubilised fraction 1 paste also containshigh levels of factor XIII and plasminogen but has low levels offibronectin.

TABLE 2 Yield of fibrinogen from solubilised fraction 1 paste Total F1Mass of paste F1 paste Total starting Fibrinogen generated extractedfibrinogen plasma YIELD Sample (g) (g) (mg) (kg) (g/kg plasma) SFP 606006.02 902.5 7476 1.22

The yield of fibrinogen from solubilised fraction 1 paste is high incomparison to solubilised heparin paste, with 1.22 g fibrinogenextracted per kilogram of plasma.

TABLE 3 Superose 6 analysis of solubilsed fraction 1 paste % Area % Area% Area % Area other Sample aggregate 1 aggregate 2 fibrinogen LMWproteins SFP 1.24 4.89 65.88 28.0

Superose 6 analysis of solubilised fraction 1 paste shows approximately65% fibrinogen monomer with low levels of aggregates but the presence oflow molecular weight proteins.

SDS-PAGE Analysis

SDS-PAGE analysis results show the presence of high molecular weightproteins under non-reducing conditions and three major bands atapproximately 40-60 kDa under reducing conditions. This profile istypical of material rich in fibrinogen.

Stability at 37° C.

The stability of solubilised fraction 1 paste was approximately 24hours. The solubilised fraction 1 paste sample clotted between 24 hrsand 32 hrs incubation at 37° C.

1.2.2 Extraction 2 Procedure

Fraction 1 paste (12.06 g) was extracted in 100.5 mL extraction buffer,centrifuged, Al(OH)₃ absorbed and Gly/NaCl precipitated.

Protein Characterisation

All samples were assayed for total protein, clottable protein, factorXIII, factor II, plasminogen and fibronectin and the yield of fibrinogenper kilogram of plasma calculated (Table 4). Size exclusion analysis wasperformed using Superose 6, the results of which are detailed in Table5.

TABLE 4 Characterisation summary Fibrinogen Total YIELD ProteinFibrinogen fibrinogen CP FXIII FII Plasm FN (g/kg Sample mg/mL mg/mL(mg) % IU/mL IU/mL μg/mL mg/mL plasma) SFP 28.11 20.42 2200 73 12.610.53 120.35 0.64 1.83 ASFP 19.43 15.00 1706 77 10.29 UD# 100.56 0.381.42 GASFP 17.63 16.26 1355 92 14.98 UD# 72.15 0.08 1.13 #undetected

Characterisation of solubilised fraction 1 paste showed extraction ofhigh levels of protein of which approximately 73% was clottable protein.This result is consistent with that obtained from Extraction I. Again,high levels of factor XIII and plasminogen and low levels of fibronectinwere also extracted. The yield of fibrinogen per kilogram of plasma wasalso high at 1.83 g/kg.

Following Al(OH)₃ absorption, factor II, which was observed to be 0.53IU/mL in the solubilised fraction 1 paste, was undetectable. Clottableprotein was observed to be 77% and the concentrations of FXIII,plasminogen and fibronectin were relatively unchanged. The yield offibrinogen per kilogram of plasma decreased by approximately 22% whichis an expected result over this step.

Gly/NaCl precipitation increased the purity of the fibrinogen to 92%clottable and removed fibronectin to negligible levels.

TABLE 5 Superose 6 analysis % Area % Area % Area % Area other LMW Sampleaggregate 1 aggregate 2 fibrinogen proteins SFP 10.85 4.78 52.47 31.90ASFP 6.32 3.20 58.86 31.62 GASFP 8.25 10.59 74.24 6.93

Superose 6 analysis of in process samples showed the purification offibrinogen over the Gly/NaCl precipitation step with an increase in thefibrinogen peak from 59% to 74% of the total area.

SDS-PAGE Analysis

SDS-PAGE analysis of solubilised fraction 1 paste showed very similarprotein composition to that generated in Extraction 1. SDS-PAGE analysisof in process samples also demonstrated the purification of fibrinogenover the Gly/NaCl step. The resolubilised Gly/NaCl precipitate samplecontains fewer high molecular weight protein bands when analysed underreducing conditions and the absence of bands at 200 kDa, 150 kDa, and 55kDa when analysed under non-reducing conditions.

1.2.3 Extraction 3 (Frozen Paste) Procedure

Fraction 1 paste (21.13 g) was extracted in 176 mL extraction buffer,centrifuged, Al(OH)₃ absorbed and Gly/NaCl precipitated. On addition ofproduct to Gly/NaCl buffer, some product clotted. Resolubilised Gly/NaClprecipitate was SD treated, applied to an ion exchange column and thefibrinogen was eluted in a salt-containing buffer.

Protein Characterisation

All samples were assayed for total protein, clottable protein, factorXIII, and factor II and the yield of fibrinogen per kilogram of plasmacalculated (Table 6). Size exclusion analysis was performed usingSuperose 6, the results of which are detailed in Table 7.

TABLE 6 Characterisation summary Total Fibrino- fibrino- Factor FactorFibrinogen Protein gen gen % XIII II g/Kg Sample mg/mL mg/mL (mg) CPIU/mL IU/ml plasma SFP clotted clotted — — clotted clotted — ASFP 17.7910.49 2029 59 6.95 UD 0.96 GASFP 12.21 10.07 836 82 clotted UD 0.40 IEX3.72 2.1 358 56 — — 0.17 column eluate #undetected

Characterisation of solubilised fraction 1 paste was not performed asthe samples clotted on thawing. One sample of resolubilised Gly/NaClprecipitate also clotted on thawing and as a result no data is availablefor factor XIII levels at this step.

Samples that were analysed demonstrated similar profiles to the previousextraction experiments. Clottable protein was approximately 60% afterAl(OH)₃ absorption and increased to greater than 80% after Gly/NaClprecipitation. Factor XIII was present at 7 IU/mL after Al(OH)₃absorption and factor II was undetectable. The yield of fibrinogen perkilogram of plasma was low with only 0.4 g/kg detected after Gly/NaClprecipitation. The GASFP was then applied to the ion exchange column topurify fibrinogen from plasminogen.

TABLE 7 Superose 6 analysis % Area % Area % Area % Area other Sampleaggregate 1 aggregate 2 fibrinogen fragments SFP clotted clotted clottedclotted ASFP 8.35 6.49 41.31 43.85 GASFP 6.42 19.59 63.56 10.42

Superose 6 analysis showed very high levels of low molecular weightproteins at the Al(OH)₃ stage. Purification of fibrinogen was again seenafter the Gly/NaCl precipitation with an increase in the fibrinogencontent from 40% to 60% of the total area.

SDS-PAGE Analysis

The first sample of solubilised fraction 1 paste was not analysed bySDS-PAGE as the sample clotted on thawing. SDS-PAGE analysis of samplesafter Al(OH)₃ and Gly/NaCl steps, under reducing and non-reducingconditions, shows the purification of fibrinogen. Analysis of the ionexchange column eluate showed that the major protein component isfibrinogen.

Stability at 37° C.

The first sample (24 hrs) of solubilised fraction 1 paste was notanalysed by SDS-PAGE as the sample clotted on thawing. The remainder ofthe solubilised fraction 1 paste stability sample clotted somewherebetween 24 hrs and 32 hrs after being left at 37° C. Analysis of theAl(OH)₃ sample showed evidence of fibrinogen breakdown at 24 hours.

After Gly/NaCl precipitation, fibrinogen was stable at 44 hours butbreakdown was evident at 144 hours (no 72 hour sample was found). When200 or 500 mM εACA was added to the resolubilised Gly/NaCl precipitate,fibrinogen was stable for greater than 240 hours.

After elution from the ion exchange column, fibrinogen was stable for atleast 208 hours (the last time point tested) without the addition of anyεACA.

1.2.4 Extraction 4 Procedure

Fresh fraction 1 paste (40.0 g) was extracted in 333 mL extractionbuffer, centrifuged, Al(OH)₃ absorbed and Gly/NaCl precipitated. εACAwas spiked into 2 sets of samples of the resolubilised Gly/NaClprecipitate at concentrations of 0 mM, 20 mM, 125 mM, 250 mM and 500 mM.One group of samples was incubated at 37° C. immediately and assessedfor stability over time. The other group of samples was stored frozen at−80° C. for 60 hours, thawed and then incubated at 37° C. for stability.Resolubilised Gly/NaCl precipitate was SD treated, applied to an ionexchange column and the fibrinogen was eluted in a salt-containingbuffer.

Protein Characterisation

All samples were assayed for total protein, clottable protein, factorXIII, factor II, and fibronectin and the yield of fibrinogen perkilogram of plasma calculated in Table 8. The pellet remaining after thefirst extraction was re-extracted with buffer containing 0.8 M NaCl.Protein characterisation of this sample and yield per kg plasma isdetailed in Table 9. Superose 6 analysis was not performed.

TABLE 8 Characterisation summary Total Fibrinogen Protein Fibrinogenfibrinogen % FXIII FII FN YIELD Sample mg/mL mg/mL (mg) CP IU/mL IU/mLmg/mL (g/kg plasma) SFP 23.69 16.09 5680 68 11.9 0.36 0.45 1.85 ASFP17.39 11.68 4472 67 9.0 UD# 0.31 1.45 GASFP 19.82 17.58 4126 89 15.0 UD#0.06 1.35 IEX Elate 8.49 8.00 704 94 NT* NT* NT* 1.07 #undetected nottested

Characterisation of in process samples showed results consistent withprevious extractions of fresh fraction 1 paste. Approximately 68%clottable protein was extracted from the fraction 1 paste. Al(OH)₃treatment reduced factor II to undetectable levels and the Gly/NaClprecipitation increased clottable protein to 89% and reduced fibronectinto negligible levels. The yield of fibrinogen per kg plasma at thesolubilised fraction 1 paste stage was 1.85 and dropped to 1.35 afterAl(OH)₃ treatment and Gly/NaCl precipitation, which is expected overthese steps. The recovery over the ion exchange chromatography step wasapproximately 76%.

Frozen GASFP was thawed and applied to the ion exchange column. Theeluate was shown to be high in clottable protein and contained lowlevels of plasminogen. The level of plasminogen in GASFP was not testedtherefore the recovery of plasminogen over the ion exchange step cannotbe calculated. However, 8 mg/mL fibrinogen and <0.2 μg/mL plasminogen inthe eluate equates to less than 1.6 μg/mL in a concentrated product of60 mg/mL. This result suggests that the ion exchange column is actingefficiently to remove plasminogen from the product.

14.13 g fraction 1 paste (remaining after extraction # 1) wassolubilised in 117.7 mL fibrinogen extraction buffer (0.8 M NaCl, pH7.3).

TABLE 9 Characterisation summary of solubilised fraction 1 paste #2Total Fibrinogen Protein Fibrinogen fibrinogen % Factor XIII Factor IIFN YIELD Sample mg/mL mg/mL (mg) CP IU/mL IU/mL (mg/mL) (g/kg plasma)SFP 26.18 17.73 2144 68 12.6 0.34 0.41 0.69Therefore the total yield of fibrinogen per kg of plasma could becalculated as 1.85+0.7 g=2.5 g fibrinogen per kg of plasma at thesolubilised fraction 1 paste stage.SDS-PAGE Analysis

SDS-PAGE analysis shows the presence of fibrinogen in all fractions.After Gly/NaCl treatment, fewer protein bands are observed underreducing conditions demonstrating the purification of the fibrinogenmolecule that is also seen by protein characterisation.

Stability at 37° C.

The resolubilised Gly/NaCl precipitate is stable for 64 hours when zeroand 20 mM εACA is added to the sample. After 64 hours the sampleclotted. With the addition of 125 mM εACA the sample is stable for 72hours but breakdown of the molecule is evident at the 100 hr time point.

Addition of 250 mM and 500 mM εACA increases the stability of theresolubilised Gly/NaCl precipitate to greater than 124 hrs, the lastsample taken.

Resolubilised Gly/NaCl samples spiked with zero or 20 mM εACA and frozenfor 60 hours at −80° C. before starting the stability trial, wereobserved to be less stable than resolubilised non-frozen precipitate.With the addition of zero or 20 mM εACA the samples were stable for 24hrs after which time they clotted compared to 64 hours in the non-frozensamples. However, the addition of 125 mM, 250 mM and 500 mM εACAincreases the stability of the fibrinogen to >96 hrs, the last timepoint taken, which did not differ significantly from 124 hours seen withthe non-frozen sample. Thus, fibrinogen is stable at −80° C. only withthe addition of at least 125 mM εACA.

Stability analysis of the ion exchange eluate was shown to be >170 hrswithout the addition of εACA.

1.2.5 Addtion of ATIII to Extraction Buffer

Fraction 1 paste (6 g), fresh and frozen, was extracted in 50 mL of theextraction buffer (±1 IU/mL ATIII), centrifuged, Al(OH)₃ absorbed andGly/NaCl precipitated. The precipitate was split in half. Half theprecipitate was stored at −80° C. and the other half was resolubilisedusing Buffer D. The resolubilised precipitate was split in half againand 0 εACA or 250 mM εACA was added. The samples were then assessed forstability at 37° C.

Protein Characterisation

Extraction 1

Fresh fraction 1 paste (6.04 g) was extracted in 50.34 g buffercontaining 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, pH 7.3. Thesolubilised fraction 1 paste was then treated with Al(OH)₃ andprecipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma was calculated (Table 10).

TABLE 10 Characterisation summary Fibrino- Total % Protein gen fibrino-clottable Fibrinogen Sample mg/mL mg/mL gen (mg) protein g/Kg plasma SFP19.56 12.14 608 62 0.85 GASFP 15.15 13.20 337 87 0.47 GASFP + 13.8911.84 313 85 0.44 250 mM εACA

Characterisation of in process samples for total and clottable proteinwas again consistent with previous results. Approximately 60% clottableprotein was extracted from fraction 1 paste which was increased to 85%clottable protein after the Gly/NaCl precipitation step. The yield waslower than previous extractions with 0.85 g fibrinogen extracted perkilogram of plasma.

Extraction 2

Fresh fraction 1 paste (5.98 g) was extracted in 49.84 g buffercontaining 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, 1 IU/ml ATIII pH 7.3.The solubilised fraction 1 paste was then treated with Al(OH)₃ andprecipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma calculated (Table 11).

TABLE 11 Characterisation summary of solubilised Fraction 1 pasteFibrino- Total Protein gen fibrino- % clottable Fibrinogen Sample mg/mLmg/mL gen(mg) protein g/Kg plasma SFP 16.15 10.48 524 65 0.74 GASFP13.63 11.79 283 86.5 0.40 GASFP + 13.76 11.86 294 86 0.42 250 mM εACA

Characterisation of in process samples generated using extraction buffercontaining 1 IU/mL ATIII was very similar to extraction buffer withoutATIII. Approximately 65% clottable protein was extracted from fraction 1paste which was increased to 85% clottable protein after the Gly/NaClprecipitation step. The yield was also lower than previous extractionswith 0.74 g fibrinogen extracted per kilogram of plasma.

Extraction 3

Frozen fraction 1 paste (6.06 g) was thawed and extracted in 50.5 gbuffer containing 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, pH 7.3. Thesolubilised fraction 1 paste was then treated with Al(OH)₃ andprecipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma calculated (Table 12).

TABLE 12 Characterisation summary of solubilised Fraction 1 pasteFibrino- Total % Protein gen fibrino- clottable Fibrinogen Sample mg/mLmg/mL gen (mg) protein g/Kg plasma SFP 12.75 8.59 416 67 0.59 GASFP 7.846.57 140 84 0.20 GASFP + 8.59 6.32 140 74 0.20 250 mM εACA

Characterisation of in process samples generated following extraction offrozen fraction 1 paste showed extraction of 67% clottable protein whichincreased to 84% following Gly/NaCl precipitation. These clottableprotein results are consistent between fresh and frozen paste startingmaterial. The yield per kilogram of plasma was lower than that extractedfrom fresh paste with less than 0.6 g fibrinogen extracted per kilogramof plasma.

Extraction 4

Frozen fraction 1 paste (6.07 g) was thawed and extracted in 50.59 gbuffer containing 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, 1 IU/mL ATIII,pH 7.3. The solubilised fraction 1 paste was treated with Al(OH)₃ andprecipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma calculated (Table 13).

TABLE 13 Characterisation summary of solubilised Fraction 1 pasteFibrino- Total Protein gen fibrino- % clottable Fibrinogen Sample mg/mLmg/mL gen (mg) protein g/kg plasma SFP 15.90 10.34 509 65 0.71 GASFP9.73 8.21 191 84 0.27 GASFP + 9.60 7.14 171 74 0.24 250 mM εACA

Characterisation of in process samples generated after extraction offrozen fraction 1 paste in buffer containing 1 IU/mL ATIII showed verysimilar results with respect to clottable protein and yield to thoseobtained from previous extractions±ATIII.

Stability at 37° C.

SDS-PAGE analysis of in process stability samples from each extractionexperiment was performed.

The stability (in hrs) of solubilised fraction 1 paste, Al(OH)₃ absorbedand Gly/NaCl precipitated samples from fresh (4° C.) and frozen (−80°C.) paste, extracted in buffers with (+) and without (−) ATM aredetailed in Table 14 below.

TABLE 14 Stability of fibrinogen (hours) 4° C. −80° C. −ATIII +ATIII−ATIII +ATIII SFP 40 74 >112 >112 ASFP 63.5 92 >112 >112GASFP >92 >112 >112 >112 GASFP + >112 >112 >112 >112 250 mM εACAAnalysis of Frozen/Thawed/Resolubilised GASFP

The frozen Gly/NaCl precipitate was thawed at 37° C. and resolubilisedusing Buffer D+100 mM εACA at 30° C. The precipitate resolubilisedwithin 30 min. Samples were then assayed for total and clottable proteinin Table 15 below and for stability at 37° C.

TABLE 15 Thawed and resolubilised Gly/NaCl precipitate ProteinFibrinogen Total fibrinogen clottable Fibrinogen GASFP (mg/mL) (mg/mL)(mg) protein (%) g/kg plasma Extraction 1 16.21 14.18 277 87 0.39 (4°C.) Extraction 2 12.80 11.18 206 87 0.29 (4° C., ATIII) Extraction 312.74 10.91 195 86 0.27 (−80° C.) Extraction 4 8.81 7.65 134 87 0.19(−80° C., ATIII)

Protein characterisation shows that the freezing of the precipitate doesnot affect the levels of clottable protein or yield of fibrinogen/kgplasma in the resolubilised Gly/NaCl precipitate.

Stability analysis of the thawed resolubilised Gly/NaCl precipitatesshowed that the fibrinogen from all extraction conditions was stable for120 hours. This result is consistent with that of the non-frozenGly/NaCl precipitates stability.

1.2.6 Addition of Heparin to Extraction Buffer

Fresh fraction 1 paste (6 g) was extracted in 50 g of the appropriateextraction buffer, treated with Al(OH)₃ and precipitated using Gly/NaClbuffer. The precipitates were then split in half. Half of theprecipitate was stored at −80° C. The other half was resolubilised usingBuffer D. The resolubilised precipitates were then split in half againand 250 mM εACA added to one half and no εACA added to the other half.The samples were then assessed for stability at 37° C.

Protein Characterisation

Extraction 1

Fresh fraction 1 paste (6.0 g) was extracted in 50.0 g of extractionbuffer containing 20 mM NaCitrate, 5 mM εACA, 0.4 M NaCl, 20 IU/mLheparin, pH 7.3. The solubilised fraction 1 paste was then treated withAl(OH)₃ and precipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma was calculated (Table 16).

TABLE 16 Characterisation summary Fibrino- Total % Protein gen fibrino-clottable Fibrinogen Sample mg/mL mg/mL gen (mg) protein g/kg plasma SFP27 18.09 974 67 1.67 ASFP 20.19 12.96 718 64 1.23 GASFP 27.29 24.11 49388 0.85 GASFP + 27.79 24.52 517 88 0.89 250 mM εACA

Characterisation of in process samples generated from extraction withbuffer containing 20 IU/mL heparin were very similar to those obtainedwith the original extraction buffer. Clottable protein was again 67%after extraction and increased to 88% after Gly/NaCl precipitation. Theyield was high with 1.67 g fibrinogen extracted per kilogram of plasma.

Extraction 2

Fresh fraction 1 paste (6.02 g) was extracted in 50.17 g buffercontaining 20 mM NaCitrate, 5 mM εACA, 0.4 M NaCl, 60 IU/ML heparin, pH7.3. The solubilised fraction 1 paste was treated with Al(OH)₃ and thenprecipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma was calculated (Table 17).

TABLE 17 Characterisation summary Fibrino- Total % Protein gen fibrino-clottable Fibrinogen Sample mg/mL mg/mL gen (mg) protein g/kg plasma SFP24.12 16.09 858 67 1.47 ASFP 19.56 13.04 707 67 1.21 GASFP 25.89 23.00440 89 0.75 GASFP + 25.64 22.82 451 89 0.77 250 mM εACA

Characterisation of in process samples from fraction 1 paste extractedwith buffer containing 60 IU/mL heparin showed results very similar tothe original extraction buffer in terms of total and clottable proteinand fibrinogen yield.

Extraction 3

Fresh fraction 1 paste (6.03 g) was extracted in 50.25 g buffercontaining 20 mM NaCitrate, 5 mM εACA, 0.4 M NaCl, 20 IU/mL heparin, 1IU/mL ATIII, pH 7.3. The solubilised fraction 1 paste was then treatedwith Al(OH)₃ and precipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma was calculated (Table 18).

TABLE 18 Characterisation summary Fibrino- Total % Protein gen fibrino-clottable Fibrinogen Sample mg/mL mg/mL gen (mg) protein g/Kg plasma SFP17.92 11.43 592 64 1.01 ASFP 12.97 7.97 419 61 0.72 GASFP 27.16 24.41439 90 0.75 GASFP + 26.27 23.25 431 88 0.73 250 mM εACA

Characterisation of in process samples from fraction 1 paste extractedin buffer containing 20 IU/mL heparin and ATIII showed results verysimilar to the those obtained with the original extraction buffer. Theyield was lower with 1 g fibrinogen extracted per kilogram of plasma.

Extraction 4

Fresh fraction 1 paste (6.01 g) was extracted in 50.09 g buffercontaining 20 mM NaCitrate, 5 mM εACA, 0.4 M NaCl, 60 IU/mL heparin, 1IU/mL ATIII, pH 7.3. The solubilised fraction 1 paste was then treatedwith Al(OH)₃ and then precipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma was calculated (Table 19).

TABLE 19 Characterisation summary Fibrino- Total % Protein gen fibrino-clottable Fibrinogen Sample mg/mL mg/mL gen (mg) protein g/Kg plasma SFP23.20 14.60 774 63 1.33 ASFP 18.42 12.13 659 66 1.13 GASFP 18.93 16.79281 89 0.48 GASFP + 19.94 17.60 305 88 0.52 250 mM εACA

Once again the results of in process characterisation of total andclottable protein from solubilise fraction 1 paste to Gly/NaClprecipitate were consistent. A significant loss of yield over theGly/NaCl precipitation stage was noted. Based on previous result,however, the presence of heparin and ATIII is not thought to contributeto the loss over this step.

Stability at 37° C.

The stability of solubilised fraction 1 paste, Al(OH)₃ absorbed andGly/NaCl precipitated samples from fresh paste, extracted in bufferscontaining 20 IU/mL or 60 IU/mL heparin, with (+) and without (−) ATIIIare detailed in Table 20 below.

TABLE 20 Stability of fibrinogen 20 IU/mL 60 IU/mL extraction −ATIII+ATIII −ATIII +ATIII buffer # (1) (3) (2) (4) SFP 23 hrs 120 hrs 23hrs >72 hrs clotted at 39 clotted at 63 ASFP 39 72 46 72 GASFP 23 63 120120 clotted at 39 clotted at 72 GASFP + 120 120 120 120 250 mM εACA

The presence of ATIII in the extraction buffer appears to increase thestability of the samples at 37° C. The presence of 60 IU/mL heparinappears to further enhance this stability to the level obtained afterthe addition of 250 mM εACA.

Analysis of Frozen/Thawed/Resolubilised GASFP

The frozen Gly/NaCl precipitate was thawed at 37° C. and resolubilisedusing Buffer D+100 mM εACA at 30° C. The precipitate resolubilisedwithin 30 min. Samples were then assayed for total and clottable proteinin Table 21 below and for stability at 37° C.

TABLE 21 Thawed resolubilised Gly/NaCl precipitate Total ClottableFibrinogen Protein Fibrinogen fibrinogen protein g/kg GASFP (mg/mL)(mg/mL) (mg) (%) plasma Extraction 1 29.08 25.86 548 89 0.94 Extraction2 28.43 25.60 555 90 0.95 Extraction 3 28.05 25.35 542 90 0.92Extraction 4 18.00 16.00 330 89 0.56

Protein characterisation shows that the freezing of the precipitate doesnot affect the levels of clottable protein or yield in the resolubilisedGly/NaCl precipitate. The yield from Gly/NaCl precipitate of Extraction4 was low but this correlated to the low yield seen in the non-frozenGASFP.

Stability analysis of the thawed resolubilised Gly/NaCl precipitatesshowed that the fibrinogen from Extraction 1 was stable for at least 36hrs (last time point), Extraction 2 was stable for at least 72 hrs (lasttime point), Extraction 3 was stable for at least 72 hrs (last timepoint), and Extraction 4 was stable for 96 hrs. This result isconsistent with that of the non-frozen Gly/NaCl precipitates stability.

1.2.7 Concentration Study

Concentration Study 1

1.5 g, 3 g, 4.5 g, 6 g, 7.5 g & 9 g of fresh fraction 1 paste wereresolubilised in 50 g of extraction buffer containing 0.8 M NaCl. Allsamples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma calculated (Table 22).

TABLE 22 Characterisation summary of solubilised Fraction 1 paste Massof paste Fibrino- Total % Fibrinogen (g) Protein gen fibrinogenclottable g/Kg Sample ratio mg/mL mg/mL (mg) protein plasma SFP #1 1.58.57 4.72 233 72 1.38 (1:33.3) SFP #2 3 12.57 8.51 433 68 1.29 (1:26.7)SFP #3 4.49 32.50 23.40 1287 72 2.56 (1:11.1) SFP #4 6.03 25.49 16.76892 66 1.32 (1:8.3)  SFP #5 7.51 30.84 21.83 1202 71 1.43 (1:6.7)  SFP#6 9.01 19.59 13.3 693 68 0.69 (1:5.5) 

Regardless of the fraction 1 paste to buffer ratio the levels ofclottable protein extracted were similar (see FIG. 1).

The yield of fibrinogen extracted per kilogram of plasma, however, isnot consistent (see FIG. 2).

The maximum yield in this experiment was obtained when a paste to bufferratio of 1:11.1 was used. After the 2 hour extraction period it wasnoted that the extraction of 1.5 g, 3 g and 4.5 g fraction 1 paste werecompletely resolubilised but the extractions of 6 g, 7.5 g and 9 gfraction 1 paste were not. This may suggest that when 4.5 g fraction 1paste in 50 ML buffer (1:11.1) is extracted completely the maximumlevels of fibrinogen are extracted. When the ratio of paste toextraction buffer ratio is increased, not all the fibrinogen present isextracted. Alternatively, the difference in yield of fibrinogen perkilogram of plasma may be due to the non-homogeneous nature of thestarting material.

Concentration Study 2

4.5 g, 9 g, 13.5 g, 18 g, 22.5 g & 27 g were resolubilised in 150 mL ofextraction buffer containing 0.8 M NaCl, 60 IU/mL heparin at 37° C. for90 minutes.

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma calculated (Table 23).

TABLE 23 Characterisation summary of solubilised fraction 1 paste #2Mass of paste Fibrino- Total % Fibrinogen g Protein gen fibrinogenclottable g/kg Sample ratio mg/mL mg/mL (mg) protein plasma SFP #1 4.517.08 4.71 706 66 1.21 (1.33.3) SFP #2 9.01 13.45 8.81 1362 65 1.17(1:26.7) SFP #3 13.50 20.00 13.10 2073 66 1.19 (1.11.1) SFP #4 18.0225.12 16.14 2633 64 1.13 (1:8.3)  SFP #5 22.51 31.99 20.31 3401 63 1.17(1:6.7)  SFP #6 27.01 35.55 22.04 3787 62 1.08 (1:5.5) 

In this experiment it was noted that after the 90 min. extraction periodall fraction 1 paste samples had been completely solubilised.

Regardless of the fraction 1 paste to buffer ratio, the levels ofclottable protein extracted were similar (see Table 23).

The yield of fibrinogen extracted per kilogram of plasma was alsounchanged over the range of paste to buffer ratios (FIG. 3). Thissuggests that the inconsistent results of the previous experiment (FIG.2) could be attributed to the non-homogeneous nature of the heparinpaste. Furthermore, this data indicates that a higher paste to bufferratio could be used in the initial extraction resulting in smallersolubilised fraction 1 paste volumes.

1.2.8 Extraction Temperature Study

Fresh fraction 1 paste (18 g) was extracted in 150 mL of buffer (20 mMNaCitrate, 5 mM εACA, 0.8 M NaCl, pH 7.3) at a ratio of 1 g:8.33 gbuffer for 2 hours at room temperature and at 37° C. Samples were takenat 30 min., 60 min., 90 min. and 120 min. throughout extraction. Thesolubilised paste was then centrifuged and another sample taken (125min).

All samples were assayed for total protein and clottable protein and theyield of fibrinogen per kilogram of plasma calculated (Table 24).

TABLE 24 Characterisation summary of solubilised Fraction 1 paste Massof Total % Fibrinogen paste Protein Fibrinogen fibrinogen clottable g/KgSample (g) mg/mL mg/mL (mg) protein plasma SFP RT - 30′ 17.99 8.97 6.581011 73 0.50 SFP RT - 60′ 13.06 9350 1460 72 0.72 SFP RT - 90′ 17.8712.88 1980 72 0.98 SFP RT - 120′ 19.62 13.80 2121 70 1.05 SFP RT - 125′20.98 15.18 2333 72 1.16 SFP 37° C. - 30′ 18.02 22.52 15.61 2568 69 1.27SFP 37° C. - 60′ 24.66 16.79 2762 68 1.37 SFP 37° C. - 90′ 27.87 19.193157 69 1.56 SFP 37° C. - 120′ 27.15 18.42 3030 68 1.50 SFP 37° C. -125′ 26.32 17.75 2920 67 1.44

Total and clottable protein extracted at RT and at 37° C. over time wasunchanged (see FIG. 4).

However, the yield calculations showed that the level of fibrinogenextracted from fraction 1 paste extracted at 37° C. was higher than thatextracted at room temperature at all time points. It was also noted thatfraction 1 paste was not completely resolubilised after 2 hr extractionat room temperature but it was completely resolubilised after 2 hrextraction at 37° C. (see FIG. 5 below).

1.2.9 Production Scale Extraction

Extraction 1

Fresh fraction 1 paste (20.0 kg) was extracted at a ratio of 1 g paste:8.33 g buffer in 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, 60 IU/mLheparin, pH 7.3, at 37° C. for 90 min.

Solubilised fraction 1 paste was treated with Al(OH)₃ and Gly/NaClprecipitation was performed. The precipitate was split into two and halfthe precipitate resolubilised in Buffer D containing 100 mM εACA and theother half frozen at −80° C. Half of this resolubilised Gly/NaClprecipitate was then treated with SD, applied to the MacroPrep column.The eluate was collected, sampled and frozen at −80° C. The other halfwas treated with SD, wet heat treated and applied to the MacroPrepcolumn. The eluate was collected, sampled and frozen at −80° C. Thefrozen Gly/NaCl pellet was thawed, resolubilised (FTR) and samples fortotal protein and clottable protein. All samples were assayed for totalprotein and clottable protein and the yield of fibrinogen per kilogramof plasma calculated (Table 25).

TABLE 25 Characterisation summary Protein Fibrinogen Total clottablePlasm. Fibrinogen Sample mg/mL mg/mL fibrinogen (mg) protein (%) (μg/mL)g/kg plasma SFP 29.09 18.15 3386790 62 1.78 ASFP 23.11 14.18 32363 611.54 GASFP 25.91 22.66 13376 87 219.6 1.27 GASFP ppt (FTR) 27.74 24.214259 87 1.35 Run 1 Eluate 11.94 10.4 770 87 0.66 0.97 Run 2 pre wetheat 1.13 0.92 691 82 0.77 Run 2 post wet heat 1.36 1.11 836 82 0.93 Run2 wet heat Eluate 2.85 2.58 263 91 0.42 0.30

Characterisation of fraction 1 paste extracted at a production scaleshowed very similar results to that extracted previously at a small labscale. Approximately 62% clottable protein was extracted at this stageat a yield of 1.78 g fibrinogen per kilogram of plasma. FollowingGly/NaCl precipitation the clottable protein again increased to greaterthan 85%, as expected. (Analysis of the frozen Gly/NaCl precipitateafter thawing and resolubilisation showed no loss of total protein,clottable protein or yield of fibrinogen per kg plasma). The materialeluted off the column was also high in clottable protein and yield. Theplasminogen level of this eluate was very low at 0.66 μg/mL whichequates to a 160 fold reduction in the level of plasminogen across thisstep.

Resolubilised Gly/NaCl precipitate that was wet heat treated and appliedto the ion exchange column was high in clottable protein before andafter the wet heat treatment. No loss of fibrinogen was seen over thewet heat treatment step. Wet heat treated material, after elution fromthe ion exchange column, was high in clottable protein but only 30% ofthe fibrinogen was recovered. Plasminogen levels were also low at 0.42μg/mL. The results of the ion exchange chromatography of GASFP and wetheat treated GASFP show that the column is acting efficiently to removeplasminogen from the product.

SDS-PAGE Analysis

All samples rich in fibrinogen as seen by the three major bands between45 and 66 kDa under reducing conditions.

Stability at 37° C.

Stability of in process samples was similar to previous findings (SeeAppendix for Stability gels). The solubilised fraction 1 paste wasstable for approximately 15 hrs. This sample clotted before 39 hrs at37° C. After Al(OH)₃ absorption, the fibrinogen molecule was stable for48 hrs and after Gly/NaCl precipitation, for >70 hrs. Following elutionfrom the ion exchange column the wet heat treated and non-wet heattreated fibrinogen was stable for at least 113 hrs.

Extraction 2

Fresh fraction 1 paste (30.0 kg) was extracted at a ratio of 1 g paste:8.33 g buffer in 20 mM NaCitrate, 5 mM εACA, 0.8 M NaCl, 60 IU/mLheparin, pH 7.3, at 37° c. for 90 min.

Total protein and clottable protein were determined and yield perkilogram of plasma calculated in Table 26 below.

TABLE 26 Characterisation of solubilised fraction 1 paste FibrinogenTotal F1 paste F1 paste Extracted Total Mass of YIELD generatedextracted F1 paste Fibrinogen fibrinogen starting plasma (g/kg (g) (g)(g) (mg/mL) (g) (kg) plasma) 59730 30000 280000 20.64 5779.2 7728 1.49

The fraction 1 paste extracted in this experiment was from the samebatch as used in the above section entitled “Concentration Study 2”. Ata small scale, the average yield of fibrinogen per kilogram of plasmawas 1.16. At a scale greater than 1,000 times this lab scale the yieldis shown to be 1.49 g/kg plasma. This further suggests that the labscale is not representative of the expected yield at production scale asa result of the non-homogeneous nature of the fraction 1 paste.

The results of the two production scale extractions show that theexpected yield of fibrinogen per kilogram of plasma is 1.5-1.8 g/kg.

1.2.10 Comparison of Fraction 1 Paste and Heparin Paste as the StartingMaterial for Fibrinogen Purification

Data gained from experiments detailed in this report is summarised inTable 27 below.

TABLE 27 Comparison of yield from fresh fraction 1 paste and heparinpaste extracted at a ratio of 6 g paste to 50 mL buffer Yield StabilityHeparin Heparin Sample Fraction 1 paste Fraction 1 paste Extracted 1.50.42 24 <24 A1(OH)3 1.32 N/A 48 N/A Gly/NaCl 0.96 0.34  >70  72 Post WetHeat 0.85 0.26  >50** 54 Post Wet Heat column 0.64* 0.23 >113  120eluate Concentrate 0.58* 0.21 >113** 120 *based on expected losses

Comparison of fraction 1 paste to heparin paste shows a significantincrease in yield of fibrinogen generated per kg plasma.

Comparison of fraction 1 paste to heparin paste in terms of stabilityshows that stability of fibrinogen increases throughout both processes.Final fibrinogen concentrate is equally stable regardless of thestarting material.

1.3 Discussion

Fibrinogen was initially extracted from fraction 1 paste at a smallscale. Experiments were performed to assess the effect of adding ATIIIand heparin to the buffer, and to assess the effect of temperature onthe extraction procedure. Results showed that the presence of heparin inthe extraction buffer increased the stability of the fibrinogen moleculeat this and subsequent steps of the process. Equal stability can also beattained by the addition of at least 125 mM εACA at the resolubilisedGly/NaCl stage.

Performing the extraction at 37° C. was shown to increase the rate ofextraction of fibrinogen and the yield of fibrinogen per kilogram ofplasma. Thus, the extraction conditions recommended for fraction 1 pasteare 20 mM Tri-sodium citrate, 0.8 M NaCl, 5 mM εACA, 60 IU/mL heparin,pH 7.3, extracted for 90 minutes at 37° C.

At a production scale, the extraction of fraction 1 paste was performedon a scale greater than 750 times that of the lab scale. In these largescale experiments, the fraction 1 paste was extracted with the optimisedbuffer (containing heparin and performed at 37° C.) and the resultantyields were 1.78 g/kg plasma and 1.49 g/kg plasma. The same batch offraction 1 paste was extracted at both lab and production scale underidentical extraction conditions and the yields obtained were 1.15 g/kgand 1.49 g/kg, respectively. With an expected yield of 1.5 g fibrinogenper kilogram of plasma at the solubilised fraction 1 paste stage, theyield from fraction 1 paste is significantly higher that that extractedfrom heparin paste (0.42 g/kg plasma).

Variation of the fraction 1 paste to extraction buffer ratio suggestedin the first small scale experiment that 4.5 g:50 mL buffer (a ratio of1:11.1) was optimal to obtain the highest yield/kg plasma. However, in asubsequent experiment performed at three times this scale and with theimproved extraction buffer, even at the highest paste to buffer ratio(1:5.5) all the fibrinogen was extracted. This result suggests thatgreater masses of fraction 1 paste may be solubilised in extractionbuffer compared to heparin paste (1:8.33) which will result in smallertotal extraction volumes.

The protein characterisation of solubilised fraction 1 paste showedsimilarities and differences to solubilised heparin paste. The amount ofclottable protein obtained from either starting material is similar atapproximately 65%. Levels of plasminogen and factor XIII were higher insolubilised fraction 1 paste than those extracted from heparin paste,however, the level of fibronectin was significantly lower. When thesolubilised fraction 1 paste was further processed using the heparinpaste method the material behaved in a similar manner to heparin pastematerial over the subsequent purification steps. Alhydrogel absorptiondemonstrated the reduction of factor II to undetectable levels thatcorrelated with an increase in fibrinogen stability at 37° C. Gly/NaClprecipitation resulted in the purification of fibrinogen to greater than80% clottable protein and the reduction of fibronectin to negligiblelevels. Ion exchange chromatography was shown to reduce plasminogen tonegligible levels in the eluate and increase the stability of thefibrinogen to approximately 120 hrs which is equivalent to heparin pasteeluate stability.

As fraction 1 paste is a by-product of another production process it isadvantageous to hold the product at this stage prior to commencing thefibrinogen manufacturing process. Heparin paste, a by-product of factorVIII concentrate can be stored frozen for up to 13 months at −80° C.without affecting the resultant levels of clottable protein onceextracted.

The stability of frozen fraction 1 paste as a starting material ispromising. After processing as far as the ion exchange column, theproduct demonstrated excellent stability (>208 hrs). In a subsequentexperiment (Section 4.5), frozen fraction 1 paste was extracted in thepresence and absence of ATIII. No handling problems were encounteredafter extraction of frozen fraction 1 paste, in either buffer, and thestability of SFP and ASFP was far greater than that observed forprevious or subsequent experiments.

Experiments were also performed to assess the possibility of holding theprocess at the Gly/NaCl precipitate stage prior to resolubilisation.Results of Gly/NaCl pellet, frozen at −80° C. and subsequently thawedand resolubilised, showed that this hold point did not compromiseproduct quality with respect to clottable protein, stability or yield.

The results presented herein show that Fraction I paste is a suitablestarting material for the purification of fibrinogen, and has thepotential to increase the yield of final product three fold compared toheparin paste.

EXAMPLE 2 Separation of Fibrinogen from Plasminogen Using Ion-ExchangeChromatography

2.1 Materials and Methods

2.1.1 Sample Preparation

A Gly/NaCl precepitate was obtained from cryoprecipitate using amodification of the methods described in Winkleman et al., 1989.Initially, the frozen resolubilised Gly/NaCl precipitate (−80° C.) wasthawed in a waterbath at 30° C. To 40 g of resolubilised Gly/NaClprecipitate was added 2.19 g of stock detergent solution and 132 mg ofTNBP. The sample was then diluted using sample dilution buffer (25 mMTris, pH 8.0) until the conductivity was below 10.5 mS/cm. Finally, thesample was filtered through a 0.8 μm membrane filter. Each sample wasprepared immediately prior to the start of each run. Failure to dilutethe sample often results in a large unbound peak i.e. some fibrinogen iseluted in the unbound.

2.1.2 MacroPrep HQ Purification

The following chromatographic conditions were used to purify theresolubilised gly/NaCl paste:

-   Bed Height—approx. 20 cm-   Column Volume—approx. 100 ml-   Flow rate—10 ml/min (˜113 cm/hr)-   Detection—UV @ 280 nm    Chromatographic Method-   Equilibration: ≧1.5 CV of MQ buffer and when conductivity    (post-column) is 90-110% of the prepared buffer.    Load Sample-   Wash: 6 CV of MQ buffer.-   Elution: ME buffer—Buffer D & 200 mM NaCl, pH 7.0-   Regeneration: 2 CV of 1 M NaCl    2.2 Results    2.2.1 Purification Using Wash Buffer (MQ—25 mM Tris, 100 mM NaCl, pH    8.0).

Duplicate runs were performed using 25 mM Tris, 100 mM NaCl, pH 8.0 asthe wash (MQ) buffer. Samples were loaded on a MacroPrep column and thecollected fractions analysed. Results are shown in Table 28.

TABLE 28 MQ-25 mM Tris 100 mM NaCl, pH 8.0 Total Clottable Non ProteinProtein Plasminogen Clottable Ratio of Fibrinogen Total VolumeCumulative % recovery % Clottable Protein Pool (mg/ml) (mg/ml) (ug/ml)Protein to Plasminogen (ml) Fibrinogen Plasminogen per fraction culm F0810a Pool 80-88 4.62 3.86 0.952 0.54 4055 90 57% 6.8% 84% 84% Tube 89 1.60.86 0.767 0.51 1121 10 58% 7.4% 54% 76% Pool 90-94 1.54 0.75 0.408 0.521838 50 64% 9.0% 49% 70% Pool 95-100 1.44 0.7 <0.2 0.49 7000 60 71% 9.5%49% 67% Pool 101-106 1.25 0.62 <0.2 0.45 6200 60 77% 9.9% 50% 65% F0910a Pool 81-86 9.75 8.73 1.68 0.45 5196 60 63% 5.9% 90% 90% Pool 87-892.64 1.88 1.15 0.47 1635 30 70% 7.3% 71% 86% Pool 90-95 1.67 0.92 0.470.46 1957 60 76% 9.5% 55% 82% Pool 96-108 1.38 0.65 <0.2 0.47 130 87%10.2% 47% 79%

The average recovery of fibrinogen was 82% and plasminogen was 10%.These figures were used as a bench mark to judge the success of anyfurther modifications to the process.

2.2.2 Addition of EACA to Load Sample.

With large scale production, not all of the samples can be processed ina single ion exchange run and hence some diluted samples were left atroom temperature while other samples are purified. It was discoveredthat the samples were breaking down during this period.

The addition of 100 mM ε-amino caproic acid, EACA (in respect to thevolume of undiluted resolubilised Gly/NaCl paste) to the sampleincreased the stability of the sample from between 0-15 hr to 15-23 hr.There were no significant changes in the chromatographic profile and therecovery of fibrinogen was 93%.

2.2.3 Addition of EACA and Lysine to Wash (MQ) Buffer.

The following wash buffers were used in the purification protocol: (i)50 mM Tris, 20 mM Lysine, 100 mM NaCl, pH 8.0 and (ii) 50 mM Tris, 20 mMEACA, 100 mM NaCl, pH 8.0. Samples were purified using the Macroprepcolumn and the collected fractions compared. Results are shown in Table29 below.

TABLE 29 Addition of EACA & Lys to MQ Buffer Protein Non ClottableClottable Protein % Protein Clottable Stability Buffer mg/ml mg/ml mg/mlas Clottable Recovery* (hrs) F14 10b Fraction 1 20 mM Lys, 50 mM Tris10.06 0.76 9.30 92% 48-71 Fraction 2 100 mM NaCl, pH 8.0 2.30 0.37 1.9484% 51.67% 48-71 Fraction 3 1.64 0.32 1.32 80% N/A Fraction 4 2.08 0.141.92 93% N/A F14 10a Fraction 1 20 mM Lys, 50 mM Tris 9.58 0.73 8.85 92%48-71 Fraction 2 100 mM NaCl, pH 8.0 1.66 0.29 1.37 83% 53.19% 48-71Fraction 3 1.71 0.18 1.52 89% N/A F15 10a Fraction 1 20 mM Lys, 50 mMTris 9.50 0.77 8.73 92% 48-71 Fraction 2 100 mM NaCl, pH 8.0 2.88 0.402.48 86% 49.11% 41-78 Fraction 3 1.63 0.32 1.31 80% N/A Fraction 4 1.890.18 1.71 90% N/A F20 10a Fraction 1 20 mM EACA, 50 mM Tris 13.27 0.8012.47 94% 71-91 Fraction 2 100 mM NaCl, pH 8.0 2.85 0.35 2.30 87% 72.12%71-91 Fraction 3 1.57 0.29 1.28 81% N/A Fraction 4 1.51 0.08 1.45 96%N/A F20 10b Fraction 1 20 mM EACA, 50 mM Tris 13.02 0.77 12.24 94% 71-91Fraction 2 100 mM NaCl, pH 8.0 2.51 0.38 2.15 86% 70.41% 71-91 Fraction3 1.81 0.29 1.32 82% N/A Fraction 4 1.52 0.05 1.47 97% N/A NB: In thesetrials EACA was not added to the sample.

With the addition of 20 mM EACA and 25 mM Tris to the MQ buffer, thestability of the collected fractions increased to between 71-91 hoursand the recovery of clottable protein was 71.3%. The longer stabilitycould possibly be attributed to the removal of plasminogen. The unboundregion of the chromatogram showed a small UV absorbance.

The average clottable protein recovery decreased to 51.3% when 20 mMlysine and 25 mM Tris were added to the MQ buffer. In thechromatographic profile, the absorbance during the wash step was largerthan that obtained using MQ. Hence it can be assumed that the additionof lysine and tris to MQ caused fibrinogen to elute in the unbound peak.The stability of the collected fractions were between 48-71 hours.

These results show that the addition of EACA and an increase of trisconcentration in MQ increased the recovery of clottable protein andstability of column eluate.

2.2.4: Varying MQ Buffer Composition

The aim of this experiment was to investigate the effectiveness of theaddition of lysine and EACA to the MQ buffer and to examine the effectof the addition of EACA to the sample. Table 30 summarises the Fraction1 results.

TABLE 30 Results of Varying Wash Buffer Composition Protein Plasmino-EACA in Buffer recovery gen Stability Sample Sample MQ plus % ug/ml HrsHPPC04 No — 77 5 24 HPPC06 No — 78 2.3 24-47 HPPC06 No Lys & Tris 480.25 47 HPPC04 Yes Lys & Tris 47 <0.5 41 HPPC06 Yes Lys & Tris 49 0.2768-93 HPPC04 Yes EACA & 68 <1.0 113-143 Tris HPPC06 Yes EACA & 790.23 >141 Tris HPPC06 Yes 25 mM Tris 77 0.8 66

These results show that when no EACA was added to the sample, and MQ (25mM Tris, 100 mM NaCl, pH 8.0) was used as the wash buffer, the averageprotein recovery of fraction 1 for HPPC04 & HPPC06 was 77% & 78%respectively. The stability was approximately 24 hours for HPPC04 and<47 hours for HPPCO6.

Where 25 mM Tris was added to the MQ buffer, the protein recovery wassimilar to that obtained using MQ but the stability of the collectedfraction was increased to 66 hours.

With the addition of lysine to the MQ buffer (50 mM Tris, 20 mM Lysine100 mM NaCl, pH 8.0), the average protein recovery of fraction 1 forHPPC06 was 48% and the stability was <47 hours. The plasminogen recoverywas reduced dramatically from 10.2% to less than 0.5%.

When EACA was added to the sample prior to loading onto the Macroprepcolumn, and the wash buffer was MQ with lysine, the protein andplasminogen recoveries were approximately the same as those obtainedwhere EACA was not added to the sample. The stability of the eluate,however, was increased from <47 hours to between 68-93 hours for HPPC06and 41 hours for HPPC04.

Where EACA was added to the samples and the wash buffer was 50 mM Tris,20 mM EACA 100 mM NaCl, pH 8.0, the protein recoveries were similar tothose obtained when using MQ i.e 68% for HPPC04 and 79% for HPPC06. Thesignificant difference was the stability of the collected fractions. Thestability was in excess of 113 hours as compared to approximately 24hours when using MQ buffer.

These results show that the wash buffer containing 50 mM Tris, 20 mMEACA 100 mM NaCl, pH 8.0 gave good results. In particular, the collectedfraction had high protein recovery, low plasminogen recovery and longstability.

2.2.5 Stability of MacroPrep Fractions, Individually and Pooled.

Previously, four fractions were collected from the MacroPrep eluate. Thefollowing experiment was designed to determine whether the fractions canbe pooled in order to increase recovery. The collected fractions wereplaced on stability both individually and pooled in the ratio as if onefraction was collected.

The purification method involved the use of 50 mM Tris, 20 mM EACA 100mM NaCl as the wash buffer and EACA was added to the samples prior toloading on the MacroPrep column. The results are shown in Table 31.

In general, Table 31 shows that the first fraction is generally morestable than the later fractions. This is most probably due to the laterfractions being considerably less concentrated than fraction 1 and not

TABLE 31 Results of Fraction Pooling Total Clottable Plasmino- ProteinClottable protein Plasminogen- Ratio Protein Protein gen Total Total Re-% Total Re- Fibrin.: Stability Sample Fraction mg/ml mg/ml ug/ml mgRecovery mg covery Clottable ug covery Plasm. Hrs F17 11a 767.3 690.62446.5 1 12.94 12.22 0.9 530.54 69% 501.02 73% 94% 36.9 1.5% 13578 >95 22.08 1.71 0.1 72.8 9% 59.85 9% 82% 3.5 0.1% 17100 46-71  3 1.12 0.86 0.1140 18% 107.5 16% 77% 12.5 0.5% 8600 <22 4 0.63 0.62 0.1 20.79 3% 20.463% 98% 3.3 0.1% 6200 <22 Unbound 1.61 0.75 0.48 54.74 7% 25.5 4% 47%16.32 0.7% 1563 N/A Unbound 1.48 0.62 0.1 50.32 9% 21.08 4% 42% 3.4 0.1%6200 N/A 2 F1 & 2 7.95 7.39 0.53 14.71 13.67 93% 0.99 13882 46-71  F1, 2& 3 3.73 3.35 0.26 18.07 16.25 90% 1.29 12649 71 F1, 2, 3, 3.29 2.960.24 18.57 16.75 90% 1.37 12271 96 & 4 F17 11b 782.4 704.2 2494.5 111.34 10.47 0.55 589.68 75% 544.44 77% 92% 26.6 1.1% 19036 46-71  2 1.871.51 0.1 65.45 8% 52.85 8% 81% 3.5 0.1% 15100 22-46  3 1.07 0.77 0.1115.56 15% 83.16 12% 72% 10.8 0.4% 7700 <71 4 0.69 0.67 0.1 22.77 3%22.11 3% 97% 3.3 0.1% 6700 <71 F1 & 2 7.54 6.88 0.37 12.59 11.48 91%0.62 18609 71-95  F1, 2 & 3 3.95 3.48 0.22 21.35 18.85 88% 1.19 15855 71F1, 2, 3, 3.47 3.08 0.20 21.99 19.47 89% 1.28 15197 71 & 4 F19 11a 753.7599.4 1953.3 1 9.02 8.45 0.1 595.32 79% 557.7 93% 94% 6.6 0.3% 84500 >952 1.7 1.44 0.1 54.4 7% 46.08 8% 85% 3.2 0.2% 14400 47 3 1 0.8 0.1 98 13%78.4 13% 80% 9.8 0.5% 8000 <23 4 0.6 0.6 0.1 19.2 3% 19.2 3% 100% 3.20.2% 6000 <23 Unbound 1.3 0.58 0.33 39 5% 17.4 3% 45% 7.5 0.4% 2320 N/AUnbound 1.44 0.6 0.1 46.08 8% 19.2 3% 42% 3.2 0.2% 6000 N/A 2 F1 & 26.63 6.16 0.10 20.28 18.85 93% 0.31 61592 <23 F1, 2 & 3 3.81 3.48 0.1023.34 21.30 91% 0.61 34796 71-95  F1, 2, 3 3.36 3.08 0.10 23.94 21.9091% 0.71 30751 95-119 & 4 F19 11b 727.7 578.7 1885.9 1 8.68 8.16 0.1581.56 80% 546.72 94% 94% 6.7 0.4% 81600 >119 2 1.76 1.76 0.1 56.32 8%56.32 10% 100% 3.2 0.2% 17600 23-47  3 1 1 0.1 97 13% 97 17% 100% 9.70.5% 10000 <23 4 0.6 0.6 0.1 19.2 3% 19.2 3% 100% 3.2 6000 <23 F1 & 26.37 6.03 0.10 19.12 18.08 95% 0.30 60267 >119 F1, 2, & 3.69 3.51 0.1022.12 21.08 95% 0.60 35133 71-95  3 F1, 2, 3, 3.25 3.10 0.10 22.72 21.6895% 0.70 30971 95-119 & 4 Samples F17 11a & F17 11b used resolubilisedGly/NaCl precipitate Batch No. HPPC04 and F19 11a & F19 11b usedresolubilised Gly/NaCl precipitate Batch No. HPPC06.because of the composition of the fractions. This was further examinedby pooling the fractions at the same ratio as they were eluted from thecolumn.

When the four fractions were pooled the stability was equivalent to thatof fraction 1. By pooling all four fractions, the recovery of proteinwas increased by approximately 25%.

2.2.6 Modification of the Elution Buffer

Results described in 2.2.5 above show that the fractions eluting fromthe MacroPrep column can be pooled. The profile of the bound fractionshows a large peak with tailing and then a second peak when the columnis washed with 1M NaCl. The second peak is the compression of thetailing due to the higher ionic strength of the 1M NaCl. It was decidedto increase the ionic strength of the elution buffer to elute fibrinogenas a single peak. The concentration of NaCl in the ME buffer wastherefore increased from 300 mM to 500 mM, 750 mM and 1M. Results areshown in Table 32.

TABLE 32 Increasing NaCl Concentration in ME Buffer Total ProteinClottable protein ME Buffer Total Total % Pool Stability Sample NaClFraction mg Recovery Cumulative mg Recovery Cumulative Clottable %Clottable Hrs F08 12a 300 mM 559.5 493.8 1 439.66 79% 79% 409.26 83% 83%93% 93% 90 2 101.64 18% 97% 88.44 18% 101% 87% 92% 72 F09 12a 500 mM615.5 543.2 1 655.6 107% 107% 599.5 110% 110% 91% 91% >100 F09 12b 500mM 615.5 543.2 1 533.9 87% 87% 478.42 88% 88% 90% 90% >100 F10 12a 750mM 654.7 577.8 1 498 76% 76% 447.6 77% 77% 90% 90% >100 2 46.9 7% 53%34.51 6% 83% 74% 88% >100 F11 12a  1 M 615.5 543.2 1 570.6 93% 93%516.24 95% 95% 90% 90% >100

The addition of an extra 200 mM NaCl to the elution buffer, ME, wassufficient to elute the fibrinogen in a single peak, with very littletailing. There was no significant difference in the characterizationresults of the collected fraction. Although, the ME buffer with 750 mMand 1 M NaCl also work, it is preferred that ME with 500 mM be used dueto the requirements of the following steps in the purification process.

2.2.7 Effect of EACA in Sample on Column Eluate Stability and Recoveries

The aim of this experiment was to compare the bound fractions eluted offthe MacroPrep column from samples (resolubilised Gly/NaCl precipitate)containing either 100 or 200 mM EACA.

Results showed that the protein recovery (93.7%, 95.4%), clottableprotein (96.0%,99.2%), plasminogen (both were <0.2 ug/ml) and stabilityresults (both were >7 days) were equivalent for the fractions collectedfrom both samples. In other words, similar results were obtained forresolubilised Gly/NaCl samples containing 100 or 200 mM εACA.

A preferred fibrinogen purification process incorporating theion-exchange chromatography method described above is shown in FIG. 6.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

References

-   Blomback and Blomback (1956). Ark Kemi. 10:415-43.-   Deutsch and Mertz. (1970). Science, 170:1095-6.-   Holm at al (1985). Thrombosis Research, 37:165-176.-   Jakobsen and Kieruif, (1973). Thrombosis Research, 3:145-159.-   Kuyas, Haeberli, Walder and Straub, (1990). Thrombosis &    Haemostasis, 64(3).439-444.-   Mosesson. (1962). Biochim. Biophys. Acta, 57:204-213.-   Robbins, Sunmnaria, Elwyn and Barlow. (1965). J. Biol. Chem,    240:541.-   Stathalis et al (1978). Thrombosis Research, 13:467-475.-   Takeda, (1966). J. Clin. Investigation, 45:103-111.-   Vuento et al (1979), Biochemistry J, 183:331-337

1. A method for purifying fibrinogen, the method comprising extractingfibrinogen from a Fraction I precipitate by admixing the Fraction Iprecipitate with an extraction buffer such that fibrinogen issolubilized in the extraction buffer, wherein the extraction buffercomprises salt at a concentration of at least 0.1 M and heparin at aconcentration of at least 10 IU/ml.
 2. A method according to claim 1wherein the concentration of salt is at least 0.4 M.
 3. A methodaccording to claim 1 wherein the salt is selected from the groupconsisting of chloride salts, phosphate salts, acetate salts and acombination thereof.
 4. A method according to claim 1 wherein the saltis NaCl.
 5. A method according to claim 1 wherein the concentration ofheparin is at least about 20 IU/ml.
 6. A method according to claim 1wherein the concentration of heparin is at least about 60 IU/ml.
 7. Amethod according to claim 1 wherein the extraction buffer furthercomprises Tri-sodium citrate at a concentration of about 20 mM.
 8. Amethod according to claim 1 wherein the extraction buffer furthercomprises at least one ω-amino acid.
 9. A method according to claim 8wherein the at least one ω-amino acid is present in the extractionbuffer at a concentration of at least 5 mM.
 10. A method according toclaim 1 wherein the extraction buffer further comprises antithrombin III(ATIII) at a concentration of at least about 1 IU/ml.
 11. A methodaccording to claim 1 wherein the extraction buffer comprises Tri-sodiumcitrate at a concentration of about 20 mM, NaCl at a concentration ofabout 0.8 M, heparin at a concentration of about 60 IU/ml and at leastone ω-amino acid at a concentration of about 5 mM.
 12. A methodaccording to claim 1 wherein the extraction buffer has a pH of about7.3.
 13. A method according to claim 1 wherein the extraction offibrinogen is performed at about 37° C.
 14. A method according to claim1, the method further comprising the step of incubating the extractedfibrinogen in solution with aluminium hydroxide followed bycentrifugation and removal of the precipitate.
 15. A method according toclaim 1, the method further comprising the step of precipitating thefibrinogen in the extracted fibrinogen solution by the addition ofglycine and NaCl.
 16. A method according to claim 15, the method furthercomprising the step of resolubilising the fibrinogen precipitate in abuffer comprising NaCl at a concentration of around 100 mM, CaCl₂ at aconcentration of around 1.1 M, Na-citrate at a concentration of around10 mM, Tris at a concentration of around 10 mM and sucrose at aconcentration of around 45 mM, with a pH of about 6.9.
 17. A methodaccording to claim 1, the method further comprising the steps of:applying the extracted fibrinogen solution to an ion exchange matrixunder conditions such that fibrinogen binds to the matrix; eluting thefibrinogen from the matrix; and optionally recovering the fibrinogenfrom the eluate.
 18. A method according to claim 17, the method furthercomprising washing the ion exchange matrix with a buffer comprising atleast one ω-amino acid prior to eluting the fibrinogen from the matrix.19. A method of purifying fibrinogen, the method comprising the stepsof: (a) extracting fibrinogen from a Fraction I precipitate by admixingthe Fraction I precipitate with an extraction buffer such thatfibrinogen is solubilised in the extraction buffer, wherein theextraction buffer comprises salt at a concentration of at least 0.1 M;(b) precipitating the fibrinogen; and (c) solubilising the fibrinogen ina solution comprising at least one ω-amino acid at a concentration of atleast 100 mM.
 20. A method according to claim 18 wherein theconcentration of salt in the extraction buffer is at least 0.4 M.
 21. Amethod according to claim 19 wherein the salt is selected from the groupconsisting of chloride salts, phosphate salts, acetate salts and acombination thereof.
 22. A method according to claim 19 wherein the saltis NaCl.
 23. A method according to claim 19 wherein the buffer furthercomprises Tn-sodium citrate at a concentration of about 20 mM.
 24. Amethod according to claim 19 wherein the extraction buffer furthercomprises heparin at a concentration of at least 10 IU/ml.
 25. A methodaccording to claim 19 wherein the at least one ω-amino acid is presentin the extraction buffer at a concentration of at least 5 mM.
 26. Amethod according to claim 19 wherein the extraction buffer comprisesNa-citrate at a concentration of about 20 mM, NaCl at a concentration ofabout 0.8 M and heparin at a concentration of about 60 IU/ml.
 27. Amethod according to claim 19 wherein the fibrinogen is precipitated instep (b) by the addition of a buffer comprising glycine and NaCl.
 28. Amethod according to claim 19 wherein the fibninogen precipitate issolubilised in step (c) using a buffer comprising NaCl at aconcentration of around 100 mM, CaCl₂ at a concentration of around 1.1M, Na-citrate at a concentration of around 10 mM, Tris at aconcentration of around 10 mM and sucrose at a concentration of around45 mM.
 29. A method according to claim 19, the method furthercomprising: (d) applying the fibrinogen solution from step (c) to an ionexchange matrix under conditions such that fibrinogen binds to thematrix; (e) eluting the fibrinogen from the matrix; and (f) optionallyrecovering the fibrinogen from the eluate.
 30. A method according toclaim 29, the method further comprising washing the ion exchange matrixwith a buffer comprising at least one ω-amino acid prior to eluting thefibrinogen from the matrix.
 31. A method according to claim 8 whereinthe at least one ω-amino acid is ε-amino caproic acid (EACA).
 32. Amethod for purifying fibrinogen, which method comprises the steps of:(a) extracting fibrinogen from Fraction 1 precipitate by admixingFraction 1 precipitate with an extraction buffer such that fibrinogen issolubilised in the extraction buffer, wherein the extraction buffercomprises at least one ω-amino acid at a concentration of at least 5 mM;(b) applying the extraction buffer from step (a) to an ion exchangematrix under conditions such that fibrinogen binds to the matrix; (c)eluting the fibrinogen from the matrix; and (d) optionally recoveringthe fibrinogen from the eluate.
 33. A method according to claim 32, themethod further comprising washing the ion exchange matrix after step (b)with a solution comprising at least one ω-amino acid.
 34. A method ofpurifying fibrinogen from a fibrinogen containing solution, the methodcomprising: (a) applying the solution to an ion exchange matrix, underconditions such that fibrinogen binds to the matrix; (b) washing the ionexchange matrix with a solution comprising at least one ω-amino acid;(c) eluting the fibrinogen from the matrix; and (d) optionallyrecovering the fibrinogen from the eluate.
 35. A method according toclaim 32 wherein the ω-amino is ε-amino caproic acid (EACA).
 36. Amethod according to claim 32 wherein the ω-amino acid is present in theextraction buffer at a concentration of between 5-500 mM.
 37. A methodaccording to claim 36 wherein the ω-amino acid is present in theextraction buffer at a concentration of between 50-500 mM.
 38. A methodaccording to claim 37 wherein the ω-amino acid is present in theextraction buffer at a concentration of about 100 mM.
 39. A methodaccording to claim 32 wherein the fibrinogen containing solution isdiluted such that the conductivity is below 10.5 mS/cm before it isapplied to the ion exchange matrix.
 40. A method according to claim 33wherein the buffer used to wash the ion exchange matrix comprises (i)Tris at a concentration of about 50 mM, (ii) a ω-amino acid at aconcentration of about 20 mM, and NaCl at a concentration of about 90mM.
 41. A method according to claim 40 wherein the buffer used to washthe ion exchange matrix has a pH of about 8.0.
 42. A method according toclaim 40 wherein the buffer used to wash the ion exchange matrix has aconductivity of about 11.1 mS/cm.
 43. A method according to claim 40wherein the fibrinogen is eluted from the matrix in a buffer comprisingabout 10 mM Tris, 10 mM citrate, 45 mM sucrose; and NaCl at aconcentration of between 200 mM to 1.0 M.
 44. A method according toclaim 43 wherein the NaCl is at a concentration of about 400-500 mM. 45.A method according to claim 43 wherein the elution buffer has a pH ofabdut 7.0.
 46. A method for purifying fibrinogen, which methodcomprises: (a) extracting fibrinogen from a fibrinogen containingmaterial by admixing the material with an extraction buffer such thatfibrinogen is solubilised in the extraction buffer, wherein theextraction buffer comprises at least one ω-amino acid at a concentrationof at least 5 mM; (b) applying the extraction buffer from step (a) to anion exchange matrix under conditions such that fibrinogen binds to thematrix; (c) washing the ion exchange matrix after step (b) with asolution comprising at least one ω-amino acid; (d) eluting thefibrinogen from the matrix; and (e) optionally recovering the fibrinogenfrom the eluate.